1 Introduction

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by pathological angiogenesis and increased angiogenic factor production, which leads to increased percolation of endothelial cells, that promotes synovitis followed by bone and cartilaginous damages, resulting in, progressive changes and destruction in joints [1]. At this time, in the year 2022, RA patients make up 1% of the world’s population [2]. Chemical medicines such as nonsteroidal anti-inflammatory medications, antirheumatic pharmaceuticals, and glucocorticoids were once the basis of RA treatment [3]. Because of their high specificity, biological antibodies have been widely used in the therapeutic treatment of a wide range of diseases in recent years, including malignancies, blood diseases, inflammation, and autoimmune diseases [4,5,6].

TNF- α (tumor necrosis factor-α) is substantially expressed in RA patients in the early stages [2]. TNF-α is a strong proinflammatory cytokine that causes local inflammation and lesion development by inducing numerous cytokines, increasing cell adhesion molecule expression, and activating blood neutrophils [2]. As a result, TNF- α can be employed as a therapeutic target for RA.

Adalimumab has an excellent specificity and affinity for TNF- α considered as first human immunoglobulin (Ig) G1 monoclonal antibody (mAb) as well as comparable terminal half-life (t1/2) (2 weeks) [7, 8]. The binding of TNF- α to its receptors is part of the inflammatory response associated with some autoimmune illnesses. Adalimumab works by neutralizing TNF’s activity and thereby preventing the cytokine’s interaction with the p55 and p75 cell surface receptors [8].

In 2002 and 2003, US Food and Drug Administration and the European Medicines Agency approved Humira (adalimumab; Abbvie Ltd.) for the treatment of rheumatoid arthritis (RA) respectively.

Now it is used to treat RA, psoriatic arthritis, juvenile idiopathic arthritis, adult Crohn’s disease, ankylosing spondylitis, pediatric Crohn's disease, and ulcerative colitis which are immune-mediated diseases. There is a wide range of study that shows similar safety, tolerability profile with the and immunosuppressive effects of the anti-TNF class, with infection, injection site reactions, headache, and rash being the most commonly reported adverse events (AEs) in adalimumab recipients [8]. Infection was the most common adverse effect, as predicted with an immunosuppressive drug, according to a global clinical database analysis of 23,458 patients, but the overall malignancy rates were average for the general population [9].

The potential structural differences in the active component that come from the biological manufacturing process need further equivalence studies, including efficacy tests in humans before biosimilar pharmaceuticals can be marketed [10]. Through a series of strictly regulated preclinical, clinical, and safety studies, A biosimilar medicinal product must compare to the authorized biological drug in terms of physicochemical, nonclinical, and clinical efficacy (known as the reference medicinal product, RMP [EU] or reference product, RP [US]) [12, 13]

Advixa is a recombinant human IgG1 mAb that is directed against human TNF- α and is developed by Incepta Pharmaceuticals Ltd, Bangladesh. It is a potential biosimilar of adalimumab and is s made in Chinese hamster ovary cells that have the same amino acid sequence as the US-licensed adalimumab reference product Humira. To confirm Advixa's basic composition, post-translational modifications, and high order structure, a comprehensive biochemical analysis was applied In comparison to US-RP. Relevant mAb production parameters, including purity, as well as product variants, were included in the similarity assessment. Advixa has the same strength as the Humira dosage formed (40 mg adalimumab per 0.4 mL deliverable solution).

A study on the administration of Advixa to healthy human participants was carried out for the first time in Bangladesh. Advixa was compared to the US-licensed Humira in terms of pharmacokinetics, safety, and tolerability. This analysis is the first step in the clinical development procedure for marketing authorization of this biosimilar product.

2 Methods

The ethical approval was taken from National Health Service Ethics Review Committee (NHERC) to conduct the trial and was carried with the Declaration of Helsinki and the principles of the International Conference on Harmonization which requires Good Clinical Practice.

2.1 Study population

The study was carried out in Mirpur in Dhaka. The study recruited 41 healthy men and women, and the sample size was calculated with the power of 80%. Participants had to weigh between 40.0 and 95 kg and have a BMI of 18.5–29.9 kg m−2 to be eligible for the study. The study protocol required that all the vital signs of participants must be within normal limits, and evaluated by physical examination. Laboratory tests including blood and urinary parameter along with a 12-lead electrocardiogram (ECG) as well as a radio imaging of the chest must be within the normal range or declared clinically non-significant. Female participants must be tested negative for pregnancy (screened and confirmed by serum and urinary β-human Chorionic Gonadotropin/ β-hCG). We excluded participants having a history of malignancy, such as lymphoma, leukemia, or skin cancer. Study participants with a positive surface antigen test for hepatitis B, antibody test for hepatitis C, and/or IgG and/or IgM for core antibody test and positive antibody test for human immunodeficiency virus at screening were excluded from the study. Participants with a positive laboratory test for drug abuse by urine examination and a positive breath alcohol test were not enrolled. Smokers who smokes more than 10 cigarettes per day or refused to refrain from smoking or using any other product that contains nicotine throughout the trial's hospital stay were excluded. Other significant exclusion criteria were history and/or current presence of atopic allergy; any present or previous local fungal infections; active or latent tuberculosis; known or suspected hypersensitivity to any drugs; history of invasive systemic fungal infections; any infection that requires hospitalization and/or anti-infectives or antibiotics within 6 months prior to trial drug administration.

The participants were completely aware of the trial's goal and were given a concept of pharmacological effects as well as the potential adverse effects of the study drug. Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. In addition, each participant received a wage loss for taking part in the study.

The study was conducted by ideSHi from June 2021 to October 2021. All the participants were screened, recruited, and data was collected from the field site situated at Mirpur, Dhaka. After enrollment, the participants were admitted to Universal Medical College and Hospital, Dhaka, which provided the in house clinical facilities for the study 12 h before dosing.

After investigational medicinal product (IMP) administration, each participant stayed for 2 days following dosing. An interim safety analysis was undertaken with data accumulated at that point after 5 participants per arm had been examined for a minimum of 2 days.

Participants were randomly assigned to groups before receiving the IMP. The randomization chart was prepared by following coded randomization. Single subcutaneous dose of 40 mg adalimumab; Advixa or Humira® was injected around 3 cm away from the navel or mid-thigh by trained nurses at the in-house clinical setting. Participants were required to remain in a relaxed state after receiving the medication and were not allowed to engage in intense activities. Blood samples were taken at each time point, and safety checks were performed as per the protocol.

Prior to dosing (-15 min) and at 4, 12, 24, 36, 48, 72 ± 2, 96 ± 2,120 ± 2, 144 ± 2, 168 ± 2, 192 ± 2, 360 ± 2, 528 ± 2, 696 ± 2, 864 ± 2, 1032 ± 2, 1200 ± 2, 1536 ± 2 h blood samples were collected for pharmacokinetic analysis (19-time points).

All blood samples were collected in Serum Separator tubes. The samples were kept upright for 30 min at room temperature followed by centrifugation for 10 min at 2000 RCF (2 ℃ − 8 ℃). Serum samples were stored at or below − 70 ℃ after aliquoting into duplicate microcentrifuge tubes.

2.2 PK assessments and endpoints

The concentrations of Advixa and Humira in the blood were measured by the ELISA method. Bound analyte, and 3,3ʹ,5,5ʹ-tetramethylbenzidine for colorimetric readout. were identified by using a TNF-coated plate, a horseradish peroxidase-conjugated anti-human IgG antibody. A plate reader was used to measure colorimetric intensity at 450 nm (detection) and 630 nm (reference) wavelengths.

Cmax, terminal half-life (t1/2), AUC from time 0 extrapolated to infinity [AUC (0)], and AUC from time 0 to the last quantifiable concentration [AUC (0, tlast)] were the primary PK endpoints.

2.3 Safety evaluation

During the trial, all adverse events (AEs) reported by the participants during the study period were well documented. Any clinically significant aberration was reported as an AE when compared to the baseline. A safety check was done for subjects who completed the 1536-h follow-up visit or withdrew from the trial early. When adverse events (AEs) occurred during the trial, the individual was tracked until the AE was resolved or stabilized. All adverse events were listed, including chest discomfort, pain, restlessness, headache, low blood pressure, swelling at the site of blood collection, vertigo, abdominal cramp, increased frequency of loose stool, upper abdominal burning sensation, itching at the site of IP administration, vomiting, burning sensation to the other thigh. The adverse events (AEs) associated with the study medication were summarized.

2.4 Statistical analysis

As the study was a parallel and double blinded study, so all the assessors along with the participants were blinded in all the stages of the assessment. A one-way ANOVA model with treatment as a fixed effect was used to investigate AUC (0), AUC (0, tlast), and the (Cmax). The PK parameters were log-transformed for analysis, and the difference in mean parameters between the two groups was calculated, then equivalency was assessed by re-expressing on the original ratio scale. For each comparison, PK equivalence was declared if the 90 percent CI for the test: reference ratio fell entirely within the 70–145 percent equivalence margin. Bioequivalence was established if all three PK metrics met the PK equivalence criterion. Other PK metrics, serum drug concentrations, and safety/tolerance data were analyzed using descriptive statistics.

3 Results

3.1 Demographics and baseline characteristics of the subjects

The study medication was given to 41 healthy subjects who were randomly selected. During the trial, 1 subject who received Humira® willingly discontinued and did not attend the follow-up visits (Fig. 1). Table 1 displays the demographic information for the 41 subjects. Subjects' basic information with Advixa was as follows: The typical age was 28.25 ± 9.53 years with an average weight of 53.07 ± 1.507 kg and an average height of 156.3 ± 7.526 cm, resulting in an average BMI of 21.65 ± 2.58 kg/m2. The basic information of subjects with Humira® was as follows: the average age was 27.29 ± 9.128 years, the average weight was 54.59 ± 1.92 kg, the average height was 156.7 ± 8.386 cm and the average BMI was 22.11 kg/m2. There was no statistically significant difference in demographic characteristics between the two groups. The inclusion criteria were met by all subjects who participated in the study.

Fig. 1
figure 1

Participant Flow chart

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Table 1 Baseline demographics and clinical characteristics
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At 19 different time intervals, the individuals' blood was drawn. ELISA was used to determine the drug concentration in the plasma. Figure 2a depicts the plasma drug concentration curve after data processing, while Fig. 2b depicts the curve after logarithmic adjustment (b).

Fig. 2
figure 2

PK analysis of adalimumab (incepta) and Humira®.After administration of the subcutaneous injection of advixa and Humira®, the Mean blood concentration–time curve; Geometric Mean (a), and log transformation (b)

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ANOVA was performed on log-transformed pharmacokinetic parameters Cmax, AUC0-t, AUC0-inf, early partial AUC, and terminal partial AUC at the α level of 0.05 for Adalimumab. As factors in the analysis of the variance model, there were sequences, and subjects nested within Sequence, period, and treatment. ANOVA and 90% confidence intervals were calculated for the ratio of geometric means of test and reference formulations using SAS system 9.4

The data in Table 2 demonstrate that there was no significant difference in plasma medication concentrations between Advixa and Humira. The average Cmax values were 7819.68 ± 3869.61 (ng/mL) and 7324.59 ± 1550.11 (ng/mL), for Advixa and Humira respectively, with a ratio of 95.56% for the modified Geometric Mean (75.20–121.44%), and the average AUC0-t values were 3,399,298.9615 ± 1,658,244.9686 and 3,004,287.9583 ± 601,639.75792 (h × ng/mL), respectively.

Table 2 After administration of subcutaneous injections, the main PK parameters of the test drug (Advixa) and reference drug (Humira)
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The findings of the equivalence determination between the test drug Advixa and reference drug Humira have been plotted in Table 3. The PK parameters were compared with the equivalence standard of 70–145%

Table 3 Results of the equivalence determination of the test drug and reference drug
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3.2 Safety evaluation

A single dose (40 mg/0.4 ml) of Adalimumab was well tolerated. Within 17 adverse events in total, almost 29.4% were in patients randomized to Advixa and 70.6% were in patients randomized to Humira®. Complaints of increased frequency of loose stool, itching over the site of IP administration, low blood pressure, and abdominal cramps were reported mostly during the entire course of the study.

Major complaints and adverse events were increased frequency of loose stool (11.76%) where 5.88% were in the Advixa group and 5.88% were due to Humira®, abdominal cramps (11.76%) were 5.88% due to Humira® and 5.88% were in Advixa group, low blood pressure (11.76%) and itching over the site of IP administration (11.76%) were entirely in Humira® group. Some minor complaints like the burning sensation of the other thigh (5.88%), vertigo (5.88%), upper abdominal burning sensation (5.88%) were entirely in Advixa group, and chest discomfort (5.88%), headache (5.88%), swelling at the site of blood collection (5.88%), vomiting (5.88%), pain (5.88%), restlessness (5.88%) were entirely in Humira® group. Among all the adverse events, 94.12% were reported by females whereas 5.88% were reported by male participants. The reported AEs were mild in severity, unlikely related to the study medication, and were resolved. No serious or significant adverse event was reported during the entire course of the study. Laboratory values were mostly found within the provided normal range but 15% of subjects reported mild anemia where 7.5% were in the Advixa group and 7.5% were in the Humira® group. In addition, 12.5% of the participants reported increased monocyte levels where 5% were in Advixa group and 7.5% were in Humira® group.

There were no significant complaints from participants. No clinically significant findings were observed during the entire course of the study. All the vital signs were within normal limits. The values which were clinically not significant were recorded in case report forms.

4 Discussion

Adalimumab biosimilar candidate Advixa is indicated for the treatment of a variety of inflammatory diseases [11]. To evaluate biosimilarity, evaluation of the PK bioequivalence of the proposed biosimilar to the reference product (RP) is required according to current guidelines. Demonstrating the PK and safety biosimilarity between Advixa and US- and EU-approved Humira was the aim of the study. In healthy subjects. For measuring PK variables, healthy volunteers are a more sensitive population group in comparison to the patient population. Selecting more homogenous healthy subjects in these cases can provide a reliable outcome as various disease-related factors that can influence the PK characteristics of study drugs can be avoided (EMEA guideline). To access equivalence between the proposed adalimumab biosimilar and the reference product, a single-dose crossover design is considered appropriate.

Biosimilars are less expensive than their reference products (RPs) and provide an opportunity to increase patient access to disease-modifying anti-rheumatic drugs (DMARDs) [12]. A number of biosimilar agents for the adalimumab reference product have been developed and evaluated in several clinical and nonclinical studies, showing similarity on the basis of structural and functional assays [13]. For several biosimilar agents such as PF-06410293, adalimumab-US, and adalimumab-EU, PK similarity was confirmed with the RP along with the structural and functional assays [14,15,16]. The present study was designed to assess the PK similarity and safety of the proposed biosimilar Advixa and US FDA-approved RP Humira in healthy Bangladeshi adult participants. This population group is considered as most sensitive to compare differences in PK and safety parameters as no immunosuppressive medications or underlying disease conditions affect the study outcome as per the ICH-GCP guidelines.

Previous biosimilarity and bioequivalence studies of Adalimumab have reported the geometric LS means ratios of primary study variables, AUCinf, AUClast, and Cmax at 90% CIs between the test and reference product were within the accepted range of 0.8–1.25 [17]. Another FKB327: Humira comparison study showed the Cmax was just outside the equivalence range of 0.8–1.25 in the upper 90% CI [18]. In the current study, the 90% CIs for the geometric mean ratios of all primary PK parameters were within the acceptance range of 70–145%. Assessment of Bio-equivalence of ‘Highly Variable Drugs products (HVDP)’ depends on its Inter Subject Coefficient of Variation. The HVDP are those whose subject variability for the PK parameter was larger than 30%. A widened acceptance range with valid clinical justification and a wider difference is considered clinically relevant for HVDP. Here, in the present study, for the parameters, Cmax, AUC0-t, and AUC0-inf, the Inter-Subject CVs (%) are larger than 30% (43.07, 49.11, and 53.39 respectively). So as per the EU guidelines, the acceptance criteria can be widened to a maximum of 69.84–143.19% (for AUC0-t, and AUC0-inf) and 72.15–138.59% (for Cmax). Moreover, the Reference Scaled approach is recommended by EMEA guidelines on the investigation of bioequivalence wherein the 90% CI can be widened in proportion to the variability of the product [13]. As the subject variability observed for all PK parameters is high, the corresponding widened BE limit has been employed for a realistic assessment of the comparative performance of the test product. The comparative assessment of secondary PK parameters like Tmax, T1/2, and Kel also showed similarity of Advixa with US- and EU-approved Humira.

In this study, 25% of participants experienced AEs, where 29.4% were in Advixa group and 70.6% were in Humira® group. No deaths or SAEs or AEs leading to discontinuation occurred during the study period. More incidences of mild to moderate AEs were reported in several bioequivalence studies [15] [18].

Limitation There is no data related to immunogenicity in this part of the clinical trial. However, the immunogenicity will be assessed in the next part of the trial in Rheumatoid Arthritis patients.

5 Conclusion

A biosimilar must have similarity to the reference biologic product in terms of structural and functional assays PK, efficacy, safety, and immunogenicity to be considered as potential [18]. In conclusion, Advixa was well tolerated by the healthy participants in this study, with a safety profile as good as that of Humira. According to the EU (Doc. Ref.: CPMP/EWP/QWP/1401/98) and WHO (WHO Technical Report Series, No. 1003, 2017 Annex 6) guidance, the 90% confidence interval of the relative mean Cmax, AUC0-t and AUC0-inf of the Advixa and Humira were found within the acceptance criteria for log-transformed data. The DSMB reviewed this Phase B study (Part A) that was done on healthy participants. Part B study on the patient with Rheumatoid Arthritis is still ongoing.